Effect of temperature was examined on the sensitivity of sandwich enzyme immunoassay for human chorionic gonadotropin (hCC) with anti-hCG Fab'-horseradish peroxidase conjugates prepared by using three different reagents (N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-l-carboxylate, glutaraldehyde and metaperiodate). The non-specific bindings of the conjugates to anti-hCC IgG-coated polystyrene balls were much lower at 20°C than at 37°C, and the specific bindings were slightly higher at 20°C than at 37°C. The lowest non-specific binding and the highest specific binding were obtained by incubation at 20°C with the maleimide conjugate. As a result, the sensitivity could be more easily improved by incubation at 20°C than at 37°C and the highest sensitivity was obtained with the maleimide conjugate. Similar results were also obtained for other macromolecular antigens such as human ferritin and α-fetoprotein.